Process and culture media for producing penicillin



Patented Apr. 27, 1948 UNITED STATE s PATENT OFFICE PROCESS AND CULTURE MEDIA FOR PRODUCING PENICILLIN Otto K. Behrens and Reuben G. Jones, Indianapolls. Ind, assignors to Eli Lilly and Company, Indianapolis, 1111]., a corporation of Indiana No Drawing. Application August 24, 1945, Serial No. 612.529

7 Claim.

This invention relates to penicillin and more particularly to improvements in the production thereof.

An object of this invention is to provide a process whereby the production and yield of penlcillin may be improved. Other objects will be The availability of penicillin has been retarded I by the circumstances of its production. The penlcillin formed. presumably as a metabolic product, by the growth of the mold under suitable conditions, has been produced slowly and in very small quantity. Furthermore, the mold has produced other products, chemically similar to penicillin, but without potent antibacterial properties. Thus the small quantity of penicillin and the presence of structurally similar compounds has rendered its isolation difficult.

One of the methods which has been employed commercially for the production of penicillin is that known as surface culture. In a common adaptation of this method a liquid nutrient medium is supplied to a large number of bottles which are supported in a substantially horizontal position. The nutrient medium is then inoculated with the Penicillium mold and the mold grows on the surface of the nutrient medium. In order to produce commercial quantities of penicillin by this method a large number of bottles is required and individual handling is necessary.

Another process which has been commercialized to some extent is that known as the bran process wherein Penicillium mold is grown on bran. The bran may be spread in thin layers on trays or may be agitated continuously in rotating drums. While the bran itself is a nutrient for the growth of the mold. it is common to associate additional nutrient materials therewith.

Probably the most widely used process at the present time for the production of penicillin is that ordinarily referred to as submerged or deep culture. Thesubmerged culture process involves the growth of Penicillium mold in an aqueous nutrient medium and accompanied customarily by agitation. when the submerged culture 2. process is carried out in small vessels such as flasks of relatively small capacity, the process is frequently referred to as the "shake" culture process because the agitation is brought about by continuously shaking the culture by suitable mechanical means. When containers of larger capacity are employed. and such containers may have capacities running into the thousands of gallons, the agitation of the liquid nutrient medium is ordinarily accomplished in part by mechanical stirring and in part by .aeration.

.which latter functions primarily to supply oxygen for the growth of the mold.

All of the above processes have been characterized in general by the slow production, and low yield, of penicillin.

By our invention the production of penicillin by a Penicillium mold of the notatum-chrysogenum group may be accelerated and the yield of penicillin increased.

According to the present invention penicillin is produced by growing a Penicillium mold of the notatum-chrysogenum group in a culture medium in the presence of the phenylacetyl compound, N-(2-aminoethyl)-pheny1acetamide, which may be represented by the formula and the acid addition salts thereof. According to aprei'erred method the Penicillium mold and the phenylacetyl compound as represented above are associated with an aqueous culture medium containing nutrient material suitable for the growth and development of the mold, and the mold is grown under penicillin-producing conditions. The phenylacetyl compbund is. incorporated in the nutrient medium in effective amount less than about 1 percent and preferably less than about 0.3 percent. The amount of material which accelerates the production of penicillin may accordingly be present in relatively small amount although over a substantial range. For example, the method may be effectively carried out by associating about 0.015 percent of N-(2- aminoethyl) -phenylacetamide and the Penicilliummold with a culture medium containing mold-growth-supporting material, and under similar conditions there may be satisfactorily employed both larger and smaller amounts of N--(2- aminoethyl) -phenylacetamide.

In another aspect there is provided by this invention, a culture medium comprising a moldgrowth-supporting aterial and a phenylacetyl compound as set forth in the next preceding paragraph. In a preferred. form the culture medium may comprise water, mold-growth-supporting material, and, in amount-effective to accelerate the production of penicillin and less than about 1 percent and preferably less than about 0.3 percent, a phenylacetyl compound as described. The amount of the compound which accelerates the production of penicillin may vary substantially throughout the range indicated above. By way of example, the culture medium may contain about 0.014 percent of N-(Z-aminoethyl) -phenylacetamlde or about 0.017 percent of N-(2 aminoethyl) -phenylacetamlde hydrochloride.

This invention will now be described in detail in its present preferred application to the submerged culture process of producing penicillin.

In the submerged culture process, the culture medium comprises water and mold-growth-supporting material. The nutrient -material may consist of ingredients known to those skilled in the art. A prominent constituent of one type of suitable nutrient material is that known as corn steep solids which is a by-product obtained in the manufacture of corn starch. Corn steep solids is a desirable material because of its low cost and its effectiveness in bringing about a relatively high yield of penicillin. On the other hand corn steep solids is of indeterminate chemical composition, some ingredients of which may make more diflicult the isolation of the penicillin produced during the growth of the mold. With a major constituent such as corn steep solids there are ordinarily associated additional ingredients known to the art such as com sugar, lactose, and salts such as calcium carbonate and zinc sulfate. The exact function of the various ingredients is not known in detail, but it is known to those skilled in the art that the combination of such ingredients does not bring about the production of penicillin when a Peniclllium mold of the notatum-chrysogenurn group is grown in such a culture medium under suitable conditions.

Another type of aqueous culture medium includes water and nutrient substances which, as contrasted with corn steep solids, are of a determinate chemical composition. Such ingredients include lactose, dextrose, acetic acid and salts such as sodium nitrate, ammonium nitrate, potassium dihydrogen phosphate, and magnesium sulfate. Compositions of this type are advantageous in that penicillin frequently is more readily separated from the other constituents of the culture medium and other products of the growth of the mold.

The mold employed for the production of penicillin is a Penicillium mold of the notatum-chrysogenum group, and illustrativly a strain of this mold suitable for the purposes of this invention is that known as strain N. R. R. L. 1976.

The phenylacetyl compound, namely N-(Z- aminoethyl) -phenylacetamide, which may be employed for the purposes of this invention to accelerate the production of penicillin, may be employed in the form of its free base and also in the form of its acid addition salts such as the hydrochloride, hydrobromide, sulfate. phosphate, succinate and maleate salts, and such salts are included within the scope of this invention.

- acetyl compound in substantial excess of the amount efleotive in promoting the maximum production of penicillin by the mold.

The phenylacetyl compound may be associated with the mold and culture medium at any suitable time. Thus the materials of the culture medium upon association in a suitable container may be inoculated with the Penicillium mold, and

' the phenylacetyl compound may. be incorporated either before or shortly after the inoculation with the mold.

The culture medium,- with the mold, and the compound accelerating the production of penicillin, should be maintained at a suitable temperature, for example in the range of 20-30 C. A range of temperature which has been found to be particularly suitable is from 2428 C. The period of time during which the mold is grown will depend upon the objective desired. Thus the mold may be grown only during the period of its maximum rate of growth. Under such conditions the mold growth may be interrupted after a period of growth of from two to three days. On the other hand the mold may be grown to obtain the maximum yield of penicillin. In such case the mold may be grown for a longer period, for example for about four or five days.

The penicillin may be separated from the culture medium in any suitable manner. For example the penicillin may be adsorbed on a surface-active carbon. Alternatively the penicillin may be extracted by means of a suitable waterimmiscible organic solvent such as amyl acetate. By well-known extractive procedures, the penicillin subsequently may be isolated in dry form as a salt thereof, for example the sodium or calclum salt.

By the practice of this invention the yield of penicillin has been substantially increased. Thus under comparative conditions the yield of penicillin has been increased from 50 to upwards of 80 percent. This range is of course illustrative and lesser or greater yields are within the contemplation of the invention depending upon the conditions under which the penicillin is produced.

Specific examples further illustrating the invention particularly with respect to the deep fiture process of producing penicillin are given Example 1 A culture medium was prepared comprising the following constituents:

Co'rn steep solids pounds 500 Com sugar ..do Lactose do-- 500 Calcium carbonate do 50 Zinc sulfate heptahydrate do 1.0 Water gallons 3,000

To the above culture medium were added about 3 pounds of N-(Z-aminoethyl) -phenylacetamide only 56 Oxford units per cc. of culture medium.

Fromtheaboveitwillbeseenthatanincrease inproductionofpenicillintotheextentoffl percent was accomplished in carrying outthe production of penicillin in accordance with this invention.

Example 2 A culture medium wasl r pared as follows:

Lactose -1 --zrams 35 Corn steep solids ..-do. 20

Zinc sulfateheptahydrate do--- 0.004

lid/50 phosphate buifer cubic centimeters- 5 Water. 1;. s --do 1,000

0.14 g. of N-(2-aminoethyl) -phenylac etamide. namely 0.014 percent on a weight-volume basis were added to the culture medium and the medium was inoculated with a Penicillium mold, strain N. R. R. L. 1076. The culture medium and mold were agitated continuously and the mold was permitted to stow for four days at 27 C. There was obtained penicillin to the extent of 196 Oxford units per cc. of culture medium.

By way of comparison, when the Penicillium mold was grown under the same conditions but in the absence of the N-(2-aminoethyl) -phenylacetamide, the yield of penicillin was 113 Oxford units per cc. of culture medium.

' In the above examples "corn steep solids" has been included as a constituent of the culture me-- dium. Corn steep solids assists in the obtaining of high yields of penicillin but due in part to the heterogeneous nature of the corn steep solids. difllculty is involved in the separation of penicillin from other constituents of-the culture medium following the growth of the mold.

In the following examples the culture medium does not contain corn steep solids and is of more precise chemical composition. The employment of such culture medium may result in the pro duction of a lower yield of penicillin. On the other hand, penicillin which is produced may be considerably more readily separated from the cul- The culture was 7 days and maintained-at a temperatu e of about 27' C. Penicillin was then found to be present to the extent of 80 Oxford units per cc. of culture By way of comparison, under the same conditions except for the absence of N-(Z-aminoethyl) -phenylacetamide, the penicillin produced was Acetic acid "do"...

only 24 Oxford units per cc. of culture medium.

7 sample 4 I Another example of a culture medium not emdylnx corn steep solids comprises the. followin: composition:

witch -c0 1,000 Lactose :rams 25.0 Dextrose -do 6.0 Sodium: nitrate do 5.0 Ammonium nitrate]. do--; 5.0 Potassium dihydroen phosphate-.do---- 1'.0 Magnesium sulfate -do 0.25 5.0

aminoethyl) -phenylacetamide hydrochloride on a weight-volume basis. was inoculated with Peni.

cillium mold strain N. B. R. L. 1976. The culture medium was mechanically agitated for 6 days and maintained at a temperature of about 27 C.

- Penicillin was produced to the extent of 85 Oxture medium following the growth of the mold.

(Adjusted to pH 6.5 with NaOH solution) N (Z-aminoethyl) -phnylacetamide was incorporated in the above culture medium to the extent of 0.014 percent and the medium inoculated ford units per cc. of-culture medium.

By way of comparison. under the same conditions except for the absence of N-(2-amlnoethyl) -phenylacetamide hydrochloride, penicillin was produced to the extent of only 54 Oxford units per cc. of culture medium. i

For the purposes'of convenience the preparation of compounds to be'employed in carrying out this invention is given below:

Preparation of N-(Z-aminoethyl) -phenulacetamide hydrochloride 164 g. of ethyl phenylacetate are mixed with moved from the reaction mixture by heating the mixture to 70 C. and subjecting it to a vacuum. preferably below 20 mm. pressure. The residue is digested with hot toluene. filtered to remove the insoluble and the filtrate evaporated in vacuo to yield a residue of the desired N-(a-aminoethyl) -phenylacetamide. The residueis dissolved in 1liter of a mixture of absolute ethyl alcohol and absolute ether and the solution saturated with dry hydrozen chloride aas. N-(z-aminoethyl) -phenylacet-= amide hydrochloride precipitates from the solution. It isseparated by filtration and purified by recrystallization from ethyl alcohol. It melts at about -141 0.

Preparation of N-(Z-azhinoethyl) -phen1llacetamide 21 x. of N-(fi-aminoethyll -phenylacetamide hydrochloride are dissolved in 50 cc. of water and a a solution of 4 g. of sodium hydroxide in 10 cc.

of water is added thereto. The mixture; is extracted several times with ether. the combined ether extracts dried with potassium carbonate and the ether removed in vacuo leaving N-(2- with a Penicillium mold, strain N. R. It. L. 1076. is aminoethyD-phenylacetainide as a colorless'oilagitated for three N,N'-di-phenylacety1ethylenediamine,

an -ass 7 What is claimed is: 4 1. The method of producing penicillin in submerged culture which comprises growing a Penicillium mold of the notatum-chrysogenum group in a culture medium in the presence of an enective amount less than about one percent of a member of the group consisting of N-ia-aminoethyli phenylacetamide represented by the formula @cm-nn-cmcnmm and acid addition salts thereof.

2. The improved method of obtaining penicillin in submerged culture which comprises providin an aqueous culture medium for the growth and development of a Penicillium mold of the notatum-chrysogenum group, associating with said culture medium a Penicillium mold of the notatum-chrysogenum group and aneffective amount less than about one percent of a member '2 of the group consisting of N-(2 -aminoethy1)- phenylacetamide represented by the formula ll @cmc-rm-cmcnmn, i

and acid addition salts thereof.

3. In the method of producing penicillin in submerged culture by growing a Penicillium mold of the notatum-chrysogenum group in association with a nutrient material, the improvement which comprises incorporating in the nutrient material, in eifective amount less than about 1 percent, a member of the group consisting of N-(z-aminoethylY-phenylacetamide represented by the formula I Q-cmd-mr-cmcnmm and acid addition salts thereof. 40

4. The improved method of producing penicillin in submerged culture which comprises providing a a culture medium containing a nutrient material, and associating with said culture medium a Penicillium mold of the notatum-chrysogenum group and about 0.014 percent of N-(2-aminoethyl) phenylacetamide represented by the formula.

' 5. A culture medium for the production of penicillin in submerged culture through the growth of a Penicillium mold of the notatum-chrysoge um at m). said culture medium comprising nutrient material and an effective amount less than about one percent of a member of the group of N-'(2-aminoethyl) -phenylacetamide represented by the formula l @cmd-na-cmcmmh 4 o ll @cmc-nn-cmcnmm and acid addition salts thereof.

'7. A culture medium for the production of peni-' cillin in submerged culture through the growth of a Penicillium mold of the notatum-chrysogenum group, said culture medium comprising water, nutrient material dispersed in said water, and about 0.014 per cent of N-(Z-aminoethyl) -phenlyacetamide represented by the formula @cmc-rm-omcnmm o'r'ro K. BEHRENS. REUBEN a. JONES.

asrnasncns CITED The following references are of record in the file of this patent:

Coghill, Monthly Progress Report No. 16. No-

5 vember 20, 1943. Distributed by Committee on Medical Research. OSBD, pages 1 and 2. 7

Pennsylvania State College, Penicillin Interim Report (-124). March 30, 1945, page 1. 

